S14G-Humanin对阿尔茨海默病转基因小鼠的治疗作用及其相关机制研究

发布时间：2018-02-13 09:04

本文关键词： 阿尔茨海默病 S14G-Humanin β-淀粉样蛋白 Morris水迷宫炎症因子免疫组织化学 ELISA 转基因小鼠方差分析　出处：《第四军医大学》2012年硕士论文　论文类型：学位论文

【摘要】：研究背景和目的：阿尔茨海默病（Alzheimer’s disease，AD）是近年来老年人最常见的神经系统变性疾病，其主要的病理学特点表现为形成老年斑、海马锥体细胞的颗粒空泡变性、神经原纤维的缠结、轴索、突触异常的断裂以及神经元的减少。该病起病比较隐匿，常伴有进行性的记忆减退和人性格的改变。到目前为止，其确切的病因和明确的发病机制尚不清楚，目前大量的研究结果显示脑内β-淀粉样蛋白（amyloid-beta protein，Aβ）在脑内聚集并且纤维化和沉积形成老年斑以及Aβ对他周围的突触和神经元的毒性作用导致AD产生和发展。S14G-Humanin（HNG）是Humanin（HN）的合成衍生物，体外研究表明该药品具有极强的神经保护功能，在动物体内初步的研究也证实HNG可有效改善侧脑室注射Aβ后所导致的认知功能障碍。然而迄今为止，HNG对于成年AD转基因小鼠已有大量Aβ斑块沉积时的治疗作用及其相关机制仍不明确，，因此，深入阐明HNG在AD发病过程中的治疗作用及其相关机制有助于探索新的AD治疗方法，并为其应用提供相应的实验基础和理论依据。本项实验我们将通过应用九月龄的阿尔茨海默病APPswe/PS1dE9转基因动物模型（APPswe/PS1dE9转基因小鼠），使用HNG疗法治疗3月，通过评定其行为学以及脑内的不同种类Aβ和胶质细胞及炎症细胞的变化进行相关分析，进一步阐明HNG疗法对已有大量Aβ斑块沉积的AD症的疗效及相关机制。研究方法：本项实验以九月龄成年APPswe/PS1dE9雌鼠和与之相匹配的常规同窝野生鼠（WT）为对象，随机分为以下4组（每组8只小鼠）：APPswe/PS1dE9+HNG治疗组，APPswe/PS1dE9+生理盐水治疗组，野生型+HNG治疗组，和野生型+生理盐水治疗组。分别给予HNG和等体积的生理盐水腹腔注射治疗3个月，然后通过Morris水迷宫，免疫组织化学，荧光染色和ELIST等方法检测小鼠的空间学习，记忆能力及不同种类Aβ和胶质细胞及炎症细胞的变化，并应用相关统计学方法深入探讨HNG在AD中的治疗作用及其相关机制。研究结果：（1）Morris水迷宫试验定位航行实验中，与WT+生理盐水治疗组和WT+HNG治疗组相比，APPswe/PS1dE9+生理盐水治疗组的潜伏期明显延长（P0.001），而与APPswe/PS1dE9+生理盐水治疗组相比，APPswe/PS1dE9+HNG治疗组的潜伏期显著缩短（P0.01）。在Morris水迷宫空间探索实验中，与WT+生理盐水治疗组和WT+HNG治疗组相比，APPswe/PS1dE9+生理盐水治疗组在目标象限停留时间明显缩短（P0.001），而与APPswe/PS1dE9+生理盐水治疗组相比，APPswe/PS1dE9+HNG治疗组在目标象限停留时间显著延长（P0.001）。（2）使用免疫组织化学和Th-S荧光染色方法分别检测APPswe/PS1dE9+生理盐水治疗组和APPswe/PS1dE9+HNG治疗组小鼠脑内Aβ斑块和纤维性Aβ斑块沉积情况。与APPswe/PS1dE9+生理盐水治疗组相比，APPswe/PS1dE9+HNG治疗组小鼠脑内皮层中Aβ斑块沉积面积及纤维性Aβ斑块沉积面积的百分比均显著减少（P0.001），海马中Aβ斑块沉积面积及纤维性Aβ斑块沉积面积的百分比也显著减少（P0.01）。（3）使用ELISA方法检测APPswe/PS1dE9+生理盐水治疗组和APPswe/PS1dE9+HNG治疗组小鼠脑内可溶性和不可溶性Aβ含量的变化情况。与APPswe/PS1dE9+生理盐水治疗组相比，APPswe/PS1dE9+HNG治疗组小鼠脑内皮层和海马中总的不可溶性Aβ含量（P0.001）、不可溶性Aβ1-40含量（P0.01）和不可溶性Aβ1-42含量（P0.001）均显著下降，而脑内皮层和海马中总的可溶性Aβ含量、可溶性Aβ_(1-40)含量和可溶性Aβ1-42含量均无显著差异（P0.05）。（4）使用ELISA方法检测APPswe/PS1dE9+生理盐水治疗组和APPswe/PS1dE9+HNG治疗组小鼠脑内APP βCTF含量的变化情况。与APPswe/PS1dE9+生理盐水治疗组相比，APPswe/PS1dE9+HNG治疗组小鼠脑内皮层和海马中APP βCTF含量均无显著差异（P0.05）。 (5)使用免疫组织化学方法检测各处理组小鼠脑内皮层和海马中反应性星形胶质细胞和活化小胶质细胞的增生情况。与WT+生理盐水治疗组和WT+HNG治疗组相比，APPswe/PS1dE9+生理盐水治疗组小鼠皮层和海马中检测到的反应性星形胶质细胞的数量以及活化小胶质的细胞数量都是明显增加的（P0.001），而与APPswe/PS1dE9+生理盐水治疗组相比，APPswe/PS1dE9+HNG治疗组小鼠皮层和海马中的反应性星形胶质细胞数量和活化小胶质细胞数量均明显减少（P0.001）（6）使用ELISA方法测量各处理组小鼠脑中炎性细胞因子IL-1β、IL-6和TNFα的含量变化并分析各组之间的差异。与WT+生理盐水治疗组和WT+HNG治疗组相比，APPswe/PS1dE9+生理盐水治疗组小鼠脑内IL-1β含量（P0.001）、IL-6含量（P0.01）和TNFα含量（P0.001）均显著增加，与APPswe/PS1dE9+生理盐水治疗组相比，APPswe/PS1dE9+HNG治疗组小鼠脑内IL-1β含量（P0.001）， IL-6含量（P0.01）和TNFα含量（P0.001）含量均显著显著减少。研究结论： 1．腹腔注射HNG可以有效改善成年APPswe/PS1dE9转基因小鼠的认知功能障碍。 2．HNG可以显著抑制成年APPswe/PS1dE9转基因小鼠脑内Aβ斑块和纤维性Aβ斑块的沉积。 3．HNG可以显著减少成年APPswe/PS1dE9转基因小鼠脑内总的不可溶性Aβ含量、不可溶性Aβ_(1-40)含量和不可溶性Aβ1-42含量。 4．HNG可以显著抑制成年APPswe/PS1dE9转基因小鼠脑内小胶质细胞和星形胶质细胞的活化和增生。 5．HNG可以显著减少成年APPswe/PS1dE9转基因小鼠脑内IL-1β、IL-6和TNFα等炎性反应因子的含量
[Abstract]:Background and purpose of the study : Alzheimer ' s disease ( AD ) is one of the most common neurodegenerative diseases in the elderly in recent years . The main pathological features of this disease are the formation of senile plaques , the vacuolation of pyramidal cells in hippocampus , the occurrence and development of neurons . Study method : In this experiment , the following four groups ( 8 mice in each group ) were randomly divided into 4 groups ( 8 mice in each group ) : the following 4 groups ( 8 mice in each group ) : the treatment group , the treatment group , the wild type + HNG treatment group , the wild type + HNG treatment group , the wild type + HNG treatment group , and the wild type + physiological saline treatment group . The spatial learning , memory capacity and the changes of different kinds of A尾and glial cells and inflammatory cells were detected by Morris water maze , immunohistochemistry , fluorescence staining and ELIST , and the treatment effect of HNG in AD and related mechanisms were discussed in detail . Results of the study : ( 1 ) In the Morris water maze test , compared with the WT + NS treatment group and WT + HNG treatment group , the latency of the treatment group was significantly shortened compared with the treatment group of WT + NS and WT + HNG ( P0.01 ) . ( 2 ) Using immunohistochemical and Th - S fluorescence staining methods , the percentage of A尾 plaque deposition area and fibrous A 尾 plaque deposition in the brain cortex of the mice were significantly decreased ( P0.01 ) compared with that in the control group , and the percentage of A尾 plaque deposition area and the fibrous A beta plaque deposition area in the hippocampus were significantly decreased ( P0.01 ) . ( 3 ) The content of soluble A尾 _ ( 1 - 40 ) and soluble A - 尾 _ ( 1 - 40 ) in the brain cortex and hippocampus of the mice were significantly lower than those in the control group ( P < 0.01 ) , but there was no significant difference between the contents of soluble A尾1 - 40 and soluble A尾1 - 42 in the brain cortex and hippocampus ( P0.05 ) . ( 4 ) There was no significant difference in the content of APP - 尾 CTF in the brain of the mice treated with the treatment group and the control group in the treatment group of APPS1d9 + NS + HNG treated by ELISA . The contents of APP - 尾 CTF in the brain cortex and hippocampus of the mice treated with APPS1d9 + HNG were not significantly different ( P < 0.05 ) . ( 5 ) The proliferation of reactive astrocytes and activated microglial cells in the cerebral cortex and hippocampus of each treatment group was detected by immunohistochemistry . The number of reactive astrocytes and the number of activated microglial cells in the cortex and hippocampus of the mice were significantly increased compared with those in the WT + NS treatment group and the WT + HNG treatment group ( P0.001 ) . ( 6 ) The contents of IL - 1尾 , IL - 6 and TNF伪 in brain of mice were measured by ELISA . The contents of IL - 1尾 , IL - 6 ( P0.01 ) and TNF - 伪 ( P0.001 ) in brain were significantly increased compared with those in WT + NS treatment group and WT + HNG treatment group . Conclusions of the study : 1 . Abdominal injection of HNG can effectively improve the cognitive impairment of the transgenic mice of the adult APGwe / PS1d9 . 2 . HNG can significantly inhibit the deposition of A尾plaques and fibrous A beta plaques in the brains of adult APPSl / PS1d9 transgenic mice . 3 . HNG could significantly reduce the content of insoluble A尾in the brain of the transgenic mice , and the content of insoluble A 尾 _ ( 1 - 40 ) and the content of insoluble A尾1 - 42 in the brains of the transgenic mice . 4 . HNG can significantly inhibit the activation and proliferation of microglial cells and astrocytes in the brains of the transgenic mice of the adult APGwe / PS1d9 . 5 . HNG can significantly reduce the levels of inflammatory response factors such as IL - 1尾 , IL - 6 , and TNF伪 in the brain of the transgenic mice of the adult APGwe / PS1d9 .

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R749.16

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